Fatty acids negatively regulate platelet function through formation of noncanonical 15‐lipoxygenase‐derived eicosanoids

Abstract The antiplatelet effect of polyunsaturated fatty acids is primarily attributed to its metabolism to bioactive metabolites by oxygenases, such as lipoxygenases (LOX). Platelets have demonstrated the ability to generate 15‐LOX‐derived metabolites (15‐oxylipins); however, whether 15‐LOX is in the platelet or is required for the formation of 15‐oxylipins remains unclear. This study seeks to elucidate whether 15‐LOX is required for the formation of 15‐oxylipins in the platelet and determine their mechanistic effects on platelet reactivity. In this study, 15‐HETrE, 15‐HETE, and 15‐HEPE attenuated collagen‐induced platelet aggregation, and 15‐HETrE inhibited platelet aggregation induced by different agonists. The observed anti‐aggregatory effect was due to the inhibition of intracellular signaling including αIIbβ3 and protein kinase C activities, calcium mobilization, and granule secretion. While 15‐HETrE inhibited platelets partially through activation of peroxisome proliferator‐activated receptor β (PPARβ), 15‐HETE also inhibited platelets partially through activation of PPARα. 15‐HETrE, 15‐HETE, or 15‐HEPE inhibited 12‐LOX in vitro, with arachidonic acid as the substrate. Additionally, a 15‐oxylipin‐dependent attenuation of 12‐HETE level was observed in platelets following ex vivo treatment with 15‐HETrE, 15‐HETE, or 15‐HEPE. Platelets treated with DGLA formed 15‐HETrE and collagen‐induced platelet aggregation was attenuated only in the presence of ML355 or aspirin, but not in the presence of 15‐LOX‐1 or 15‐LOX‐2 inhibitors. Expression of 15‐LOX‐1, but not 15‐LOX‐2, was decreased in leukocyte‐depleted platelets compared to non‐depleted platelets. Taken together, these findings suggest that 15‐oxylipins regulate platelet reactivity; however, platelet expression of 15‐LOX‐1 is low, suggesting that 15‐oxylipins may be formed in the platelet through a 15‐LOX‐independent pathway.


| INTRODUC TI ON
Long-chain polyunsaturated fatty acids (PUFAs) are shown to be protective against cardiovascular diseases 1,2 ; however, the mechanism of this effect is not well understood. PUFAs have been shown to regulate and alter platelet function through their metabolism to bioactive oxylipins by the two main oxygenases, cyclooxygenase (COX) and lipoxygenase (LOX). 3 Platelets express COX-1, whose inhibition by non-steroidal anti-inflammatory drugs (NSAIDS) is thought to be a primary reason for the observed decrease in platelet reactivity. 2,4 Regarding the role of lipoxygenases in platelets, 12-LOX is highly expressed and plays an important role in regulating platelet activation. 5,6 However, the presence of 15-LOX in the platelet and whether it is required for the formation of 15-LOX-derived oxylipins   from dihomoγ-linolenic acid (DGLA), the source of these molecules is poorly defined. 7,8 Lipoxygenases could potentially generate these molecules; however, studies have indicated that these 15-oxylipins are generated by COX. 2,9 Interestingly, the role of these 15-oxylipins in platelet biology is also controversial.
While 15-HETrE, 15-HETE, and 15(S)-hydroxyeicosapentaenoic , from eicosapentaenoic acid (EPA), have been shown to inhibit platelet reactivity, 9,10 other studies have observed a proaggregatory effect of 15-HETE on platelet function. 11,12 With respect to their mechanism of action, oxylipins can inhibit platelet function by increasing cyclic adenosine monophosphate (cAMP) levels via Gα s -coupled receptors, or binding intracellular nuclear receptor, such as peroxisome proliferator-activated receptors

| Isolation of human platelets
All research involving human subjects was carried out in accordance with the Declaration of Helsinki and approved by the University of Michigan Institutional Review Board. Prior to blood collection, written informed consent was obtained from all subjects in this study.
Platelets were pelleted from the column flow-through by centrifugation following treatment with acid citrate dextrose and apyrase, as described above.

Significance Statement
We demonstrate that in vitro treatment with 15-HETrE, 15-HETE, or 15-HEPE negatively regulates platelet reactivity through inhibition of platelet intracellular signaling that attenuates platelet activation and reduces 12-LOX activity. These effects are mediated, in part, through the activation of PPARs.

| Statistical analysis
Two-tailed paired t test, one-way, and two-way analysis of variance

| 15-Oxylipins inhibit agonist-induced platelet aggregation
We have shown that human platelets treated with DGLA were able to form 15-HETrE ex vivo ( Figure

| 12-LOX product profile and kinetics with C20 PUFAs and 15-oxylipins substrates
The

| 12-LOX allosteric and hypoxic regulation of epoxidation
Oxylipins have been previously determined to dose dependently affect the ratio of di-oxygenation: epoxidation products, 29,31 due to allosteric regulation of enzyme mechanism. In order to determine if 15-HpEPE could also affect the ratio of LOX products, the product profile was assessed in solutions ranging from 1 to 20 μM of 15-HpEPE. Increased concentrations of 15-HpEPE reduced epoxide formation from 84% at 1 μM 15-HpEPE to 58% at 20 μM (Table 3), indicating that 15-HpEPE is also a 12-LOX allosteric regulator that affects secondary product formation.

| Ex vivo platelet incubation with 15-oxylipins
The in vitro assays indicate that 12-LOX reacts slowly with 15-oxylipins under in vitro conditions and, therefore, the  platelets, and is consistent with the amount of di-oxylipin produced when 14(S)-hydroperoxydedocosahexaenoic acid (14-HpDHA) was added to platelets. 29 Second, only 1/10 of the reduced alcohol oxylipin products were observed compared to the hydroperoxide oxylipin products, which is consistent with the in vitro kinetics, where the alcohol oxylipins were observed to be poorer substrates than the hydroperoxides. Finally, the 8,15-product from epoxide degradation was the major di-oxylipin species when 15-HpEPE was added to platelets ( Table 4), suggesting that the hydroperoxide oxylipin is a viable 12-LOX substrate in the platelet.
The aforementioned data suggest that treatment of platelets

| 15-HETrE and 15-HETE inhibit platelet activation via unique PPARs
Independent of direct effects on oxygenases, oxylipins have additionally been proposed to reduce platelet activation through either the initiation of Gα s -coupled receptor signaling or stimulation of PPARs. 1 The inhibitory effects of the Gα s signaling pathway proceed through cAMP-dependent PKA activation. 40 In platelets, the major substrate of PKA is VASP serine 157 (S157). 41 To determine if 15-HETrE or 15-HETE regulates platelet function in this manner, VASP phosphorylation was measured in platelets treated with oxylipins (10 μM) to assess their ability to initiate Gα s signaling. VASP (S157) phosphorylation did not increase in platelets incubated with 15-HETrE or 15-HETE compared to either DMSO or 12-HETE, a negative control ( Figure 5A). As expected, platelets treated with either forskolin, a direct adenylyl cyclase agonist, or 12-HETrE, a 12-LOX oxylipin that signals through a Gα s -coupled receptor, 20 had enhanced VASP phosphorylation.
Platelets express all three PPAR isoforms (α, β, and γ) and activation of any of these isoforms inhibit platelet function through

| 15-LOX expression in human platelets
In this study, we demonstrated that leukocyte-depleted platelets treated with DGLA prior to agonist-induced activation generated 15-HETrE ( Figure 1B) as observed with 12-HETE and 12-HETrE, but rather shifts the isoform of PPAR that is activated.
Oxylipin inhibition of platelet function through negative feedback on the production of pro-aggregatory oxylipins has been previously shown. 24 In agreement with previous studies, 10 Previously, our group has demonstrated that treatment with DGLA increased levels of the 12-LOX-derived oxylipin, 12-HETrE, in platelets. 20 In accordance with previous findings, 8,9 we observe is highly expressed in leukocytes, 2 it is reasonable to consider that in whole blood, platelet reactivity might be partially regulated by a transcellular mechanism between platelets and leukocytes through the formation of 15-oxylipins. Therefore, this leukocyte-platelet interaction could regulate clot formation and thus have clinical implications in atherothrombotic diseases through inhibition of platelet activity and thrombosis. Wrote or contributed to the writing of the manuscript.

ACK N OWLED G M ENT
We thank Amanda Prieur for recruiting subjects and performing blood draws.

CO N FLI C T O F I NTER E S T S TATEM ENT
Dr. Holinstat is a consultant and equity holder and consultant for Veralox therapeutics and Cereno Scientific. All other authors declare no competing interests for the work reported in this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
All subjects participated voluntarily and received a small compensation. The participants provided their written informed consent to participated in this study. The Declaration of Helsinki was adequately addressed, and the study was approved by the University of Michigan Institutional Review Board.